The present invention relates to liquid chromatography instrumentation, and more particularly to a method and apparatus for absorbance detectors allowing multiple pathlength flow cells.
Absorbance detectors are useful in high performance liquid chromatography (HPLC). Broad spectrum or bandwidth limited light is directed through a sample, and then measured at the chosen analytical wavelengths by a detector, such as a photodetector. In a traditional instrument, light traverses a fixed distance (path length) through the sample. The instrument""s photodetector signal is measured when the analyte sample concentration is zero (I0) and when the analyte is present (I). Absorbance (A), a dimensionless number expressed in absorbance units, a.u., is calculated from log(I0/I) and displayed as the instrument output. Absorbance is proportional to the product of pathlength (b) and concentration (c)-Beer""s Law. The constant of proportionality can be found from a calibration experiment using known analyte concentrations, thus enabling unknown concentrations to be measured.
If pathlength is expressed in cm and concentration in moles/L, the proportionality constant is called the molar absorbtivity (xcex5) with units cmxe2x88x921 (moles/L)xe2x88x921.
Since xcex5 varies with wavelength for any analyte, the instrument includes a monochromator, filters, a diode array spectrograph or, in the case of the infrared a Fourier transform interferometer, so that absorbance is measured at specific wavelengths.
The range of concentrations of an analyte which can be measured in such an instrument is limited. At the low end, the minimum detectable change in absorbance is set by the base line noise on the absorbance output, a value which varies from wavelength to wavelength and from instrument to instrument. A well-designed UV absorbance detector for HPLC can detect an absorbance change in the region of 10 to 20xcexca.u. An upper limit of concentration measurement is reached when the relationship between absorbance and concentration becomes significantly nonlinear. This typically occurs when absorbance exceeds 1 to 2 a.u. The upper absorbance limit is usually the result of stray light or inadequate spectral resolution. The upper absorbance limit varies with wavelength and from instrument to instrument, and is reduced if the solvent or HPLC mobile phase absorbs.
The analyte concentration range as used herein is defined as the ratio of the maximum to minimum concentration. Because of the above, it is limited to about five orders of magnitude. If the goal is to quantitate to 1%, the analyte concentration range (assuming comparable molar absorptivities among components) cannot exceed three decades. This can result in the need for more than one HPLC run with different sample injections in order to quantitate major components and trace impurities in a sample mixture.
Beer""s Law shows that lower concentrations can be detected if the cell pathlength is increased, and higher concentrations will fall within the linear absorbance range if the pathlength is decreased. But changing the pathlength will in itself neither increase nor decrease the concentration range.
Further, cell detector design is close to limits imposed by the physics of available components (light sources, photodetectors etc.), the constraints on cell volume required to maintain chromatographic resolution, and market-driven requirements of spectral range and resolution. It is already a challenge to build detectors and chromatographic systems which do not have some spurious source of noise in excess of the value set by physics. Even if the theoretical noise could be reduced by improved design, such significantly lower noise may not be realized in practice.
Long pathlength light-guiding flowcells offer a way to increase concentration sensitivity for a given baseline noise. Unfortunately, the high concentration limit, set by the detector""s linear absorbance range, is reduced by the same amount, so that the concentration range remains the same. As previously noted, if the mobile phase absorbs, the concentration range will actually be less with a longer cell.
Wide concentration range is a very important attribute of a detector. It enables major components and trace impurities (as in a drug formulation) to be quantitated in a single injection. In this application of an analytical scale separation, a wide concentration range, using larger injections of sample, is more useful than the ultimate in low detection limits.
Providing dual pathlengths to measure a wider range of concentrations has been attempted with varying degrees of success. U.S. Pat. No. 5,214,593 (Magnussen) discloses a method of using multi-pathlength flow cells using at least two light beams and at least two photo detectors. Light which passes through a standard length sample cell impinges on a sample photodiode and light which passes through a (shorter) reference cell impinges on a reference photodiode. At low sample concentrations the detector behaves like a conventional dual beam instrument with very little sample-induced change in the reference photodiode signal. At high concentrations, the sample photodiode signal falls to zero and the instrument""s reference beam behaves like a single beam detector. The signal processing electronics select the photodiode output appropriate to the concentration range. Each pathlength has its own light beam and detector. The two detectors produce signals which are processed separately and then combined. This requires expensive redundancy in equipment, and would be particularly expensive to implement using photodiode array detectors.
In fact, as shown in FIGS. 1, 2 and 3 of Magnussen the use of three detectors is preferred, with the third detector serving as a reference. This can greatly increase the cost of the apparatus.
Further, Magnussen requires the apparatus to be especially designed to perform multi-pathlength flow cell analysis. The instrument must be built with multiple light beams, multiple light detectors, and multiple channels for detector signal processing. The implementation taught by Magnussen can not be used in existing single-pathlength systems.
U.S. Pat. No. 4,120,592 (Flemming) discloses a multiplex optical analyzer apparatus. The apparatus uses multiple path lengths with a single light source and detector. The light beam only passes through one cell pathlength at a time, by the action of a light spectrum filter wheel and spectrum sensitive beam splitters, as shown in FIG. 1 of Flemming. Thus Flemming only measures one pathlength at a time, requiring extra time for analyzing samples. Also, similar to Magnussen, Flemming requires a specially designed system to perform multi-pathlength flow cell analysis.
The present invention provides a dual or multi-path length flow cell wherein light from the different sample pathlengths is combined and impinges on the same photodetector. This implementation can be used with photodiode array detectors which conventionally have only a single cell and a single photodiode array.
According to a dual pathlength embodiment of the present invention, a flow cell is provided in which a portion of the light traverses a long pathlength and the balance of the light traverses a much shorter pathlength. Light entering the flow cell is split between the two paths and then recombined to be passed to the photodetector. The dual path flow cell can in principle replace the single path cell in a spectrometer or detector. At low analyte concentrations, changes in light transmission in the long path portion of the cell are sensitive to small changes in concentration. The slope of the calibration curve, A versus c, is high in this region. At high concentrations the long path becomes opaque and the detector response depends on light in the short path. The slope A versus c is proportional to pathlength and is much less when the short path length dominates. At intermediate concentrations, the slope transitions between these two regions.
Because the overall curve of A versus c is nonlinear, a much wider range of concentrations falls within the limited absorbance range of the detector. Once the instrument response versus concentration of a particular analyte is characterized, the instrument can be used for quantitative analysis. The length ratio of the two paths can be quite large, on the order of 100:1. Other than the form of the algorithm to compute sample concentration discussed in detail hereinafter, there is no change in the operation of the detector or spectrometer using the dual path length flow cell according to the invention. The concepts according to the invention are related herein to absorbance detectors for high performance liquid chromatography (HPLC), but are equally applicable to bench-top spectrophotometers.
Features of the invention include provision of a multipath flow cell that increases the range of analyte concentration that can be measured in an absorbance detector by combining light which has traversed at least two different sample pathlengths. The quantity and cost of related equipment is minimized according to the invention in that a single detector is used to receive light that passes through each of the varied length multiple paths.
The present invention provides the ability to use multipath flow cells in a traditional single path system thereby providing expanded capablities of present equipment.